Sample carrier based on a porous film with metal oxide particles, the production and utilization thereof, especially for selective detection of phosphorylated/sulphatized biopolymers

ABSTRACT

The present invention relates to a porous film comprising metal oxide particles, to a sample holder using the film, to a method of preparation for the sample holder, to the application of the sample holder as well as to a method of detecting phosphorylated/sulphated biopolymers, specifically peptides/proteins.

The present invention relates to a porous film comprising metal oxideparticles, to a sample holder using the film, to a method of preparingthe sample holder, to the application of the sample holder as well as toa method of detecting phosphorylated/sulphated biopolymers, specificallypeptides/proteins.

Phosphorylation and de-phosphorylation processes on biopolymers,specifically proteins, play an important role in the regulation ofcellular functions. They take in influence, inter alia, on growth, onmetabolism and on the gene expression or dominance. The recognition ofthe phosphorylation pattern of metabolism and proteome analysis maycontribute to a better understanding of the complex regulatory networksand signal transmission systems in a cell. The complete analysis of thephosphorylation pattern of a biologic sample is a great challenge inbiochemistry or bioanalysis because the phosphorylated biopolymers,particularly proteins, are present in very low concentrations, inaddition to a great number of other bio molecules. Mass spectrometry isone of the most important analytical methods of characterisingbiopolymers, in particular proteins and peptides. It is an analyticalmethod for the identification of compounds, for checking them for purityand for the exact determination of the qualitative and quantitativecomposition of a sample as well as of the mass of a compound. In allforms of mass spectrometry, the formation of ions of the species to bestudied is required, which are subsequently subdivided and detected in adetector device. Hence, each mass spectrometer comprises three modules,i.e. an ion source, a device for ion separation as well as a device forion detection. Depending on the type of ion generation, a distinction ismade between electron pulse ionisation mass spectrometry (El), chemicalionisation mass spectrometry (CI), Fast Atom Bombardment massspectrometry (FAB) and matrix-assisted laser desorption/ionisation massspectrometry (MALDI). The matrix-assisted laser desorption ionisationmethod (MALDI), linked up with a mass spectrometer, is the method forhigh-throughput analysis of biopolymer, particularly protein andpeptide, mixtures. Due to the low concentration ofphosphorylated/sulphated biopolymers, particularly proteins/peptides,complete signal suppression may occur in such a mixture, which meansthat the detection of these compounds of often impossible. There isaccordingly a demand in this field for a method of detectingphosphorylated/sulphated biopolymers, particularly peptides/proteins,which is highly responsive to these substances and selective. Inparticular, there is a demand for devices and methods facilitating theperformance of matrix-assisted laser desorption ionisation massspectrometry (MALDI mass spectrometry) and rendering them selective anddistinctly more responsive to the aforementioned substances.

The problem of the present invention hence resides in the facilitationof the detection of phosphorylated/sulphated biopolymers, specific allypeptides/proteins, also in complex mixtures. It is the objective of thepresent invention, in particular, to provide a device and a methodfacilitating the detection of phosphorylated/sulphated biopolymers,specifically peptides/proteins, by means of MALDI mass spectrometry andenhancing the responsiveness of the detection.

These problems are solved by a sample holder for application in MALDImass spectrometry, comprising:

a substrate,

a porous film comprising metal oxide particles, which is applied on thesubstrate.

The metal oxide particles are preferably selected from the groupincluding titanium dioxide, zirconium dioxide, niobium oxide, aluminiumtitanium oxide, tungsten zirconium oxide, hafnium dioxides, tungstenoxide, tin dioxide, lead oxide, lead dioxide, germanium dioxide andgallium oxide (TiO₂, ZrO₂, NbO, Al₂TiO₅, W₂ZrO₈, TiZrO₄, HfO₂, WO₃,SnO₂, PbO, PbO₂, GeO₂ and Ga₂O₃).

According to a particularly preferred embodiment, the metal oxideparticles are particles of titanium dioxide, zirconium dioxide and/ortitanium zirconium oxide or of a mixture of the aforementionedcompounds.

In one embodiment, the film presents an average pore size of <50 nm,preferably a mean pore size in the range from 1 nm to 25 nm, and mostpreferably a mean pore size in the range between 1 nm and 10 nm.

The film should preferably have a thickness of 0.1 μm to 10 μm,particularly a thickness in the range from 2 to 4 μm and most preferablya thickness of roughly 3 μm.

According to one embodiment, the substrate consists of glass or coatedglass. In one embodiment, the glass is conductive glass or glass with aconductive coating. According to a preferred embodiment, the substrateconsists of glass coated with indium tin oxide (ITO).

In one embodiment, the substrate consists of aluminium, aluminium withan aluminium oxide layer, especially anodically oxidised aluminium(electrolytic oxidation).

According to one embodiment, the porous film provided on the substratecomprises additionally:

a MALDI matrix.

In one embodiment, the MALDI matrix comprises a substance selected fromthe group 2,5-dihydroxy benzoic acid, 3,5-dimethoxy-4-hydroxy cinnamicacid, α-cyano-4-hydroxy cinnamic acid, ferulic acid, 2,4,6-trihydroxyacetophenone.

The term “MALDI matrix” as used herein is to be understood in the sensethat it denotes any substance suitable as matrix for performance ofMALDI mass spectrometry. Examples of such substances are mentionedhereinbelow.

The term “biopolymers” as used herein is to be understood to denotespecifically proteins, peptides, nucleic acids, lipids andlipo-polysaccharides.

In one embodiment, the film provided on the substrate is present only ondefined areas specifically envisaged to this end and covers them whilstother areas therebetween are left free of the film.

Such “structuring” may be achieved by means of methods and processesknown in this field, for example screen-printing techniques,spin-coating techniques, dip-coating techniques, doctor blade methods,drop-casting techniques, each with or without an appropriate dot matrixmask/dot matrix mask sheet presenting the pattern to be applied anddesired for the metal oxide film.

In one embodiment, the sample holder moreover comprises:

One or several samples to be analysed, which are applied on the film andwhich are each presumed to contain one or several substances ofinterest, it being preferred that the sample(s) contain(s) substancesthat are selected from the group including nucleic acids and proteins.

It is most preferred that the proteins are phosphorylated and/orsulphated.

The term “substance of interest” as used herein denotes a substancewhose detection is envisaged.

In particular, the term “substance of interest” denotes a phosphorylatedand/or sulphated biopolymer.

It should be emphasised here that the precise type and nature of thesubstrate in the inventive sample holder is not essential as long as thesuitability of the substrate to hold the inventive film is ensured. As aconsequence, the problems of the present invention are also solved by aporous film, independently of a sample holder, which film comprisesmetal oxide particles, with the film presenting the aforementionedfeatures in preferred embodiments. In one embodiment, the inventive filmis applied on a substrate as specified above.

The problems of the present invention are furthermore solved by theapplication of an inventive film or sample holder, respectively, for thedetection of phosphorylated/sulphated biopolymers, specificallypeptides/proteins from peptide/protein mixtures, with the detectionbeing preferably carried out by means of matrix-assisted laserdesorption ionisation mass spectrometry (MALDI).

The problems of the present invention are also solved by a method ofselective detection of phosphorylated/sulphated biopolymers,specifically peptides/proteins from peptide/protein mixtures, comprisingthe following steps of operation:

-   -   providing a sample holder according to the present invention,    -   providing a sample which is presumed to contain        phosphorylated/sulphated biopolymers, specifically        peptides/proteins, alone or in combination with other        biopolymers, specifically peptides/proteins, and applying the        sample on the sample holder,    -   performing MALDI mass spectrometry.

Moreover, the problems of the present invention are solved by a methodof preparing a sample holder for application in MALDI mass spectrometry,with the sample holder including a substrate and a film applied on thesubstrate and including metal oxide particles, which method comprisesthe following steps of operation:

-   -   preparing a sol from a metal oxide,    -   inducing gel formation, for example by restriction and/or        thermal treatment,    -   applying the gel on a substrate,    -   drying and subsequent tempering at 200-600° C., preferably 300        to 450° C., most preferably at roughly 400° C., for a period of        30 minutes to 180 minutes, preferably 30 minutes to 60 minutes,        most preferably for roughly 45 minutes.

The metal oxide is preferably selected from the group including titaniumdioxide, zirconium dioxide, niobium oxide, aluminium titanium oxide,tungsten zirconium oxide, titanium zirconium oxide, hafnium dioxide,tungsten oxide, tin dioxide, lead oxide, lead dioxide, germanium dioxideand gallium oxide.

In one embodiment, the film presents a mean pore size of <50 nm,preferably in the range from 1 nm to 25 nm, most preferably in the rangebetween 1 nm and 10 nm.

The problems underlying the present invention are equally solved by asample holder that can be prepared by a method according to the presentinvention, it being preferred that the sample holder comprises a porousfilm of metal oxide particles, with the film presenting a mean pore sizeof <50 nm, preferably from 1 to 25 nm, most preferably in the range from1 to 10 nm.

The problems of the present invention are furthermore solved also by anapplication of a sample holder according to the present invention forthe selective detection of phosphorylated/sulphated biopolymers,specifically peptides/proteins, with the detection being preferablyperformed by means of MALDI mass spectrometry.

The problems underlying the present invention are also solved by amethod of preparing a sample for MALDI mass spectrometry, using aninventive sample holder, with the method including the following stepsof operation:

-   -   providing an inventive sample holder,    -   applying a sample on the metal oxide film of the sample holder,        with the sample being presumed to contain        phosphorylated/sulphated biopolymers, specifically proteins,    -   one or several washings steps for washing the metal oxide film,    -   applying a phosphate-containing medium on the metal oxide film        of the sample holder,    -   applying a MALDI matrix on the metal oxide film of the sample        holder.

It is possible to perform a drying step, as required, after theindividual steps. Examples of phosphate-containing media arephosphate-containing buffer solutions such as sodium phosphate,potassium phosphate, ammonium phosphate at an appropriate pH level.According to an alternative to the separate application of aphosphate-containing medium and the application of a MALDI matrix, it isalso possible that the MALDI matrix as such comprises phosphate groups.Washing in the washing step/washing steps may be performed byapplication of or rinsing with water, diluted acids, buffer solutions,etc.

The term “MALDI matrix” as used herein is to be understood to denote anysubstance suitable as a matrix for performing MALDI mass spectrometry.Examples of this are mentioned in the following.

The invention for which this application is filed relates to a sampleholder or to a method of selective and highly responsive detection ofphosphorylated/sulphated biopolymers, respectively, specificallypeptides/proteins in complex peptide/protein mixtures, by means of MALDImass spectrometry. The invention permits surprisingly a rapid processingof the samples (roughly 30 minutes), extremely small sample volumes (0.5μl), a strong selectivity and a high responsiveness, whilst it issuitable for a high sample throughput. The invention involves thepreparation of sample holders from meso-porous metal oxide films for thedetection of phosphorylated and/or sulphated bio molecules and furnishesprotocols for the immobilisation, purification and release of the biomolecules on the sample holder. Not intending to be invariablyconcentrated on a particular mechanism, the inventors presently startout from the fact that the basis of the method is the selectiveimmobilisation of the phosphorylated/sulphated bio molecules incorrespondence with their affinity to the meso-porous film and thesubsequent release and analysis in a MALDI mass spectrometer. The filmconsists of particulate metal oxides in the nano range, particularlytitanium dioxide and zirconium dioxide, which are produced by means of asol/gel process.

The term “pore size” as used herein refers to the interstitial spacepresent between the particles positioned in the complex, indicatingtheir average dimensions. The term “meso-porous” as used herein meansthat the average pore size is in the range below 50 nm, preferablywithin the range from 1 to 25 nm, most preferably in the range from 1 to10 nm. The term “particulate metal oxides in the nano range” denotesmetal oxides having a particulate character, with the average particlesize ranging from 1 to 30 nm.

MALDI sample holders are distributed by the leading manufacturers ofmass spectrometers (Applied Biosystems, Waters/Micromass,BrukerDaltoniks). These sample holders serve, on the one hand, to conveythe sample into the mass spectrometer and/or to purify or concentratethe sample. A selective immobilisation of phosphorylated/sulphated biomolecules is not achieved by means of commercially available sampleholders. The Cyphergen Company offers Protein Arrays on the basis ofmetal oxides. Here, the pore size exceeds 100 nm, however, so that it isnot meso-porous. Moreover, selectivity in terms ofphosphorylated/sulphated biopolymers, specifically peptides/proteins, isnot described either.

The Immobilised Metal Affinity Chromatography (IMAC) process is equallycommon and also commercially available (Sigma/Aldrich, Pierce). In thismethod, the phosphorylated peptides are selectively linked by means ofiron cations or gallium cations and are subsequently released again. Thedisadvantage of this process resides in the aspect that rathersubstantial sample volumes are required and that immobilisation takesplace “off line”, i.e. by means of a separate column/cartridge and thatthe sample is subsequently applied on a MALDI sample holder. With thisapproach, it is possible that some sample material is lost and that theresponsiveness is reduced.

Moreover, studies have become known from the group around Suzuki et al.(“2D-LC for Selective Extraction of Phosphopeptides using TitaniaPrecolumn“; Poster Abstract, 31^(st) ASMS Conference, Jan. 8-12, 2003,Montreal, Canada), in which the use of titanium dioxide is described asnew packing material for the HPLC analysis for the selectivepurification of phosphorylated compounds. Moreover, the use of apre-purifying step by means of titanium dioxide HPLC in a columnspecifically provided to this end is described, with the subsequentanalysis by means of mass spectrometry of this pre-purified sample beingproposed. The direct application of titanium dioxide films in the massspectrometer is not taken into consideration.

Now, reference is made to the drawings in which

FIG. 1 is an exemplary schematic view of a sample holder made of ITOglass with a metal oxide film applied in islets, with the diameter ofthe individual “islets” amounting to 1 to 3 mm;

FIG. 2 shows the application of the sample holder according to FIG. 1 ina metal holder of a MALDI mass spectrometer (Voyager DE), with a recessbeing cut out of the metal holder, which presents the size of thecontours of the sample holder.

FIG. 3 is a photograph of a holder for an inventive sample holder forapplication in MALDI time-of-flight mass spectrometry (MALDI-TOF);

FIG. 4 shows a photograph of another holder for an inventive sampleholder for use in MALDI quadrupole ion-trap mass spectrometry underatmospheric pressure (AP/MALDI-QIT);

FIG. 5 illustrates the mass spectrum of a tryptical digestion of betacasein without application of the inventive sample holder, and

FIG. 6 shows the mass spectrum of a tryptical digestion of beta caseinwith application of an inventive sample holder.

In the following, the invention will now be explained in more details bythe specific examples discussed for explanation only, rather than for alimitation of the invention.

EXAMPLE 1

Production of one embodiment of a meso-porous metal oxide film

25 ml of titanium tetra-isopropoxide are mixed with 10 ml of isopropanolin a dropping funnel and slowly dripped into 200 ml of water while beingstirred. Subsequently, 5 ml to 100 ml of concentrated nitric acid areadded (other organic acids may, however, be equally used, such as aceticacid, formic acid, etc.) and cooked for 6 hours with refluxing. Theisopropanol so formed is withdrawn. The resulting sol is then processedin an autoclave at 180 degrees Celsius or 18 hours. The resulting sol isthen reduced to 15% w/w titanium oxide in a rotary evaporated at 40° C.and 30 mbar, mixed with 20% Carbowax and stirred for 24 hours. Theviscous solution is spread on a substrate, e.g. made of glass coatedwith indium tin oxide (ITO). The substrate was structured by means of anadhesive sheet into which holes of 2 mm diameters, for example, had beenpunched. Substrate structuring may be performed by any method suitableto this end. Such methods are common to those skilled in the art.Exemplary methods that may be employed and result in a sample holderincluding areas with and areas without a metal oxide film, are asfollows: screen-printing process, spin-coating process, dip-coatingprocess, doctor blade method, drop-casting techniques, each with orwithout an appropriate dot matrix mask/dot matrix mask sheet presentingthe pattern to be applied and desired for the metal oxide film.

When the solution has been dried the sheet is removed. Then, the glasssubstrate is tempered at 400° C. for 45 minutes. In this manner,meso-porous metal oxide films are produced which have a diameter of 2 mmand a thickness of 2 micrometers. After cooling, the ITO glass is cutinto pieces having a size of 3×2 cm.

The concentration of the phosphorylated/sulphated proteins/peptidesduring application of the sample takes place via an inventive metaloxide film. Immediately after the application of the sample, appropriate(different) washing solutions are used for washing, which are appliedand removed again after an appropriate period, with the washingsolutions serving to aim at two different objectives, without theinventors intending to be bound to a specific mechanism: 1. washing outthe non-combined non-phosphorylated proteins/peptides, 2. Loosening upthe bonding of the phosphorylated proteins/peptides by the addition of aphosphate-containing buffer, with the phosphates contained in the bufferremoving the combined phosphorylated proteins/peptides at least partlyout of their bond to the metal oxide film and rendering them thusaccessible to the desorption ionisation process. As a matter of fact, itwas surprising to find that the application of a phosphate-containingbuffer onto the metal oxide film enhances distinctly the responsivenessin the subsequent mass spectrometry operation. Subsequently, a matrixcommon for the MALDI technique (for instance 2,5-dihydroxy benzoic acid,3,5-di-methoxy-4-hydroxy cinnamic acid, ferulic acid, 2,4,6-trihydroxyacetophenone, α-cyano-4-hydroxy cinnamic acid (HCCA), is applied. As analternative of the separate application of a phosphate-containing bufferand a MALDI matrix it is equally possible to apply aphosphate-containing MALDI matrix that acts not only as a matrix butinduces also the aforementioned “phosphate exchange”. The introductionof the sample holder so prepared and provided with the sample into themass spectrometer is carried through, for instance, by means of amodified metal support for the MALDI-TOF Voyager DE device of theApplied Biosystems Company. A recess in the size of the ITO glass wasmilled into the metal support. The glass is placed into the recess andcontacted with metal tongues (cf. FIGS. 1 and 2 for a schematic view andFIGS. 3 and 4 for photographs of sample holders actually produced). FIG.1 illustrates a glass (1) with ITO coating, on which a metal oxide filmis provided in an “islet” arrangement, with the individual spots(islets) 2 of the metal oxide film presenting a mean diameter of 1 to 3mm.

FIG. 2 is a schematic view of a holder (3) of a MALDI mass spectrometer(or Voyager DE from Applied Biosystems) into which a recess (4) havingthe size of the glass with ITO coating according to FIG. 1 has beenmilled. Moreover, FIG. 2 shows a metal tongue (5) for contacting theinventive sample holder (e.g. the glass with ITO coating from FIG. 1).FIGS. 3 and 4 illustrate a holder for an inventive sample holder, whichhas been prepared and actually employed in practical operation; FIG. 3shows a holder used in practical operation for MALDI Time-of-flight massspectrometry (MALDI-TOF) whilst FIG. 4 illustrates a holder used forMALDI quadrupole ion trap mass spectrometry at atmospheric pressure(AP/MALDI-QIT). The metal oxide film applied in the form of islets canbe seen in the form of circles on the glass in both figures.

EXAMPLE 2

For the detection of phosphorylated peptides by means of MALDI massspectrometry the following technique may be employed, for example:

The concept of this technique is as follows: the sample to be analysed,which is presumed to contain phosphorylated peptides(sulphated peptides,is applied on an inventive MALDI sample holder (with a metal oxide film,which concentrates the phosphorylated peptides/proteins selectivelywhilst other proteins/peptides, which are not phosphorylated/sulphated,can be removed by means of suitable washing solutions. The result is asample holder enriched with phosphorylated/sulphated proteins/peptides,which may be subsequently used directly in MALDI mass spectrometry.Different buffers may be used for the washing steps following theapplication of the sample, depending on the type, volume and origin ofthe applied sample. Then a matrix common for the MALDI technique isapplied, which will be referred to here as “MALDI matrix”.

The following protocol may be employed, for instance, for the successfuland highly selective detection of phosphorylated peptides:

Protocol for the detection of phosphorylated peptides

1 μl of the sample solution is pipetted onto a film prepared accordingto Example 1

Wait for 15 minutes

Remove supernatant solution by means of a pipette

Wash three times with 2 μl of water

Wash three times with 2 μl mM of acetic acid

Pipette 1 μl of 100 mM ammonium dihydrogen phosphate onto the film

Wait for 15 minutes

Pipette MALDI matrix onto the film (for instance: 2,5-dihydroxy benzoicacid, 3,5-dimethoxy-4-hydroxy cinnamic acid, ferulic acid,2,4,6-trihydroxy acetophenone, α-cyano-4-hydroxy cinnamic acid)

Let it dry and measure by means of a MALDI mass spectrometer.

As an alternative, it is possible to apply aphosphate-containing/phosphate group-containing MALDI matrix onto thefilm instead of the separate application of a phosphate buffer.

EXAMPLE 3

FIGS. 5 and 6 illustrate a mass spectrum of a tryptical digestion ofbeta casein, which is obtained without (FIG. 5) and with (FIG. 4) theuse of an inventive sample holder (trypt. digestion 15 pmol/μl, HCCAmatrix 10 mg/ml, 1 μl of the sample). The peaks associated with phosphorpeptides (phosphorylated peptides) are marked by an asterisk. Therespective peaks in FIG. 5 are extremely small and are clearlysuperimposed by other peaks of non-phosphorylated peptides. By contrast,FIG. 6 shows the three highest peaks as peaks based on phosphorylatedpeptides. This means that the use of an inventive sample holder resultsin a highly selective and highly responsive detection of phosphorylatedpeptides.

As the sample holder is a component of the MALDI spectrometer or of theassociated process, respectively, expensive and troublesome preliminarypurification steps in separate devices are no longer required and, so tospeak, “on-line” immobilisation, purification, concentration and releaseof phosphorylated/sulphated peptides/proteins takes place, which rendersthe inventive device or the inventive method, respectively, bestsuitable for a high-throughput analysis by means of MALDI massspectrometry.

The features of the invention, which have been disclosed in theforegoing description, in the claims as well as in the drawings, may beessential for the implementation of the invention in its differentembodiments, both individually and in any combination whatsoever.

1. Sample holder for application in MALDI mass spectrometry, comprising:a substrate a porous film present on the substrate and comprising metaloxide particles.
 2. Sample holder according to claim 1, characterised inthat the metal oxide particles are selected from the group includingtitanium dioxide, zirconium dioxide, niobium oxide, aluminium titaniumoxide, tungsten zirconium oxide, hafnium dioxides, tungsten oxide, tindioxide, lead oxide, lead dioxide, germanium dioxide and gallium oxide(TiO₂, ZrO₂, NbO, Al₂TiO₅, W₂ZrO₈, TiZrO₄, HfO₂, WO₃, SnO₂, PbO, PbO₂,GeO₂ and Ga₂O₃).
 3. Sample holder according to claim 1, characterised inthat the film has a mean pore size of <50 nm.
 4. Sample holder accordingto claim 3, characterised in that the film has a mean pore size in therange from 1 nm to 25 nm.
 5. Sample holder according to claim 4,characterised in that the film has a mean pore size in the range from 1nm to 10 nm.
 6. Sample holder according to claim 1, characterised inthat the film has a thickness in the range from 0.1 pm to 10 pm. 7.Sample holder according to claim 6, characterised in that the film has athickness in the range from 2 to 4 pm.
 8. Sample holder according toclaim 7, characterised in that the film has a thickness of roughly 3 μm.9. Sample holder according to claim 1, characterised in that thesubstrate consists of glass or coated glass.
 10. Sample holder accordingto claim 9, characterised in that the glass is a conductive glass or aglass with a conductive coating.
 11. Sample holder according to claim 9,characterised in that the substrate consists of glass coated with indiumtin oxide (ITO).
 12. Sample holder according to claim 1, characterisedin that the porous film applied on the substrate moreover comprises. 13.Sample holder according to claim 12, characterised in that the MALDImatrix comprises a substance selected from the group: 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxy cinnamic acid, a-cyano-4-hydroxycinnamic acid, ferulic acid, 2,4,6-trihydroxy acetophenone.
 14. Sampleholder according to claim 1, characterised in that the film applied onthe substrate is present only at defined areas specifically envisaged tothis end, covering same, whereas other ranges therebetween are left freeof the film.
 15. Sample holder according to claim 1, furthermorecomprising: one or several samples to be analysed, which are applied onthe film and which is or are presumed to contain one or severalsubstances of interest.
 16. Sample holder according to claim 15,characterised in that said one or several sample(s) contain(s)substances selected from the group including nucleic acids and proteins.17. Sample holder according to claim 16, characterised in that theproteins are phosphorylated and/or sulphated.
 18. Method of selectivedetection of phosphorylated/sulphated biopolymers, specificallypeptides/proteins from peptide/protein mixtures, comprising thefollowing steps of operation: providing a sample holder according to thepresent invention, providing a sample which is presumed to containphosphorylated/sulphated biopolymers, specifically peptides/proteins,alone or in combination with other biopolymers, specificallypeptides/proteins, and applying the sample on the sample holder,performing MALDI mass spectrometry.
 19. Method of preparing a sampleholder for application in MALDI mass spectrometry, with the sampleholder comprising a substrate and a porous film applied on the substrateand including metal oxide particle, which method comprises the followingsteps of operation: preparing a sol from a metal oxide, inducing gelformation, for example by restriction and/or thermal treatment, applyingthe gel on a substrate, drying and subsequent tempering at 200-600° C.,preferably 300 to 450° C., most preferably at roughly 400° C., for aperiod of 30 minutes to 180 minutes, preferably 30 minutes to 60minutes, most preferably for roughly 45 minutes.
 20. Method according toclaim 19, characterised in that the metal oxide is selected from thegroup including titanium dioxide, zirconium dioxide, niobium oxide,aluminium titanium oxide, tungsten zirconium oxide, hafnium dioxides,tungsten oxide, tin dioxide, lead oxide, lead dioxide, germanium dioxideand gallium oxide (TiO₂, ZrO₂, NbO, Al₂TiO₅, W₂ZrO₈, TiZrO₄, HfO₂, WO₃,SnO₂, PbO, PbO₂, GeO₂ and Ga₂O₃).
 21. Method according to claim 19,characterised in that the film has a mean pore size of <50 nm. 22.Method according to claim 21, characterised in that the film has a meanpore size in the range from 1 nm to 25 nm.
 23. Method according to claim22, characterised in that the film has a mean pore size in the rangefrom 1 nm to 10 nm.
 24. Sample holder suitable for preparation accordingto a method according to claim
 19. 25. Sample holder according to claim24, comprising a porous film of metal oxide particles, with the filmhaving a mean pore size of <50 nm, preferably in the range from 1 to 25nm, most preferably in the range from 1 nm to 10 nm.
 26. Application ofa sample holder according to claim 1 for selective detection ofphosphorylated/sulphated biopolymers, specifically peptides/proteins.27. Application according to claim 26, with detection being performed bymeans of MALDI mass spectrometry.
 28. Method of preparing a sample forMALDI mass spectrometry, using a sample holder according to claim 1,with the method comprising the following steps of operation: Providing asample holder according to claim 1, Applying a sample on the metal oxidefilm of the sample holder, which is presumed to containphosphorylated/sulphated biopolymers, specifically peptides/proteins,Washing the metal oxide film in one or several washing operations,Applying a phosphate-containing medium onto the metal oxide film of thesample holder, applying a MALDI matrix onto the metal oxide film of thesample holder.
 29. Application of the method according to claim 28 forperforming MALDI mass spectrometry.
 30. Method of preparing a sample forMALDI mass spectrometry, using a sample holder according to claim 24,with the method comprising the following steps of operation: Providing asample holder according to claim 24, Applying a sample on the metaloxide film of the sample holder, which is presumed to containphosphorylated/sulphated biopolymers, specifically peptides/proteins,Washing the metal oxide film in one or several washing operations,Applying a phosphate-containing medium onto the metal oxide film of thesample holder, applying a MALDI matrix onto the metal oxide film of thesample holder.